Electrochemical Biosensor Designed to Distinguish Tetracyclines Derivatives by ssDNA Aptamer Labelled with Ferrocene

Abstract

Controlling food safety and preventing the growing spread of antibiotics into food products have been challenging problems for the protection of human health. Hence, the development of easy-to-use, fast, and sensitive analytical methods for the detection of antibiotics in food products has become one of the priorities in the food industry. In this paper, an electrochemical platform based on the ssDNA aptamer for the selective detection of tetracycline has been proposed. The aptasensor is based on a thiolated aptamer, labelled with ferrocene, which has been covalently co-immobilized onto a gold electrode surface with 6-mercaptohexan-1-ol. The changes in the redox activity of ferrocene observed on the aptamer-antibiotics interactions have been the basis of analytical signal generation registered by square-wave voltammetry. Furthermore, the detection of tetracycline-spiked cow milk samples has been successfully demonstrated. The limits of detection (LODs) have been obtained of 0.16 nM and 0.20 nM in the buffer and spiked cow milk, respectively, which exceed the maximum residue level (225 nM) more than 1000 times. The proposed aptasensor offers high selectivity for tetracycline against other structurally related tetracycline derivatives. The developed biosensor characterized by simplicity, a low detection limit, and high reliability shows practical potential for the detection of tetracycline in animal-origin milk.

Keywords: antibiotics; aptamer; cow milk samples; electrochemical biosensor; ferrocene; tetracycline.

Figure 1

Chemical structures of tetracyclines.

Scheme 1

(A) Scheme of aptasensor preparation dedicated to TET detection. (B) Scheme of carrying out an experiment for the determination of TET using an electrochemical aptasensor.

Figure 2

(A) Cyclic voltammograms recorded for the constructed aptasensor at different scan rates: 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 mV/s. (B) Linear relationship between the scan rates and redox peak currents observed for Fc. Potential measured vs. Ag/AgCl.

Figure 3

Results of (A) repeatability, (B) reproducibility, and (C) stability studies obtained for the gold electrodes modified with Au-S-Apt-Fc/MCH.

Figure 4

Example of square-wave voltammograms registered for gold electrodes modified with Au-S-Apt-Fc/MCH upon interaction with (A) TET, (B) OTC, and (C) DOX. Black curve (1) before and next curves after 1 h interaction with antibiotics at particular concentrations: 0.1 nM (red, 2), 0.5 nM (blue, 3), 1.0 nM (pink, 4), 5.0 nM (green, 5), and 10.0 nM (violet, 6). (D) Relative intensity of current (ΔI) vs. concentration of (●) TET, (♦) OTC, and (■) DOX for gold electrodes modified with Au-S-Apt-Fc/MCH.

Scheme 2

General scheme of ferrocene-based electrochemical aptasensors “signal-on” working principle.

Figure 5

(A) SWV responses registered for different concentrations of TET prepared in pre-treated cow milk: 0.1nM (red, 3), 0.5nM (blue, 4), and 1.0nM (pink, 5). Black curve—registered in buffer (1), dashed curve—registered in the cow milk matrix (2), and next curves after 1 h interaction with TET. (B) The calibration curves of aptasensor recorded for TET in (●) buffer solution and (▲) spiked cow milk with the concentration range of 0.1–1.0 nM (n = 4).

Figure 6

Selectivity of the prepared aptasensor in cow milk spiked with TET, DOX, OTC, and mixture of antibiotics: TET, DOX, and OTC with the same concentration of 0.1 nM (n = 4).